The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)–A0201 epitope, is a validated target for T cell–based immunotherapy. Using phage display technology, we discovered a fully human “T cell receptor–like” monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1-and HLA-A0201–restricted manner, with high avidity [dissociation constant (Kd) = 0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome–positive leukemia in nonobese diabetic/severe combined immunodeficient γc−/− (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.
BACKGROUND
The transcription factor, WT1, is highly overexpressed in malignant pleural mesothelioma (MPM) and immunohistochemical stains for WT1 are used routinely to aid in its diagnosis. Using computer prediction analysis we designed analog peptides derived from WT1 sequences by substituting amino acids at key HLA-A0201 binding positions. We tested the safety and immunogenicity of a WT1 vaccine comprised of four class 1 and class 2 peptides in patients with thoracic neoplasms expressing WT1.
METHODS
Therapy consisted of six subcutaneous vaccinations administered with Montanide adjuvant on weeks 0, 4, 6, 8, 10, and 12, with 6 additional monthly injections for responding patients. Injection sites were pre-stimulated with GM-CSF (70mcg). Immune responses were evaluated by DTH, CD4 T-cell proliferation, CD8 T-cell interferon gamma release, intracellular cytokine staining, WT1 peptide MHC-tetramer staining, and cytotoxicity against WT1 positive tumor cells.
RESULTS
Nine patients with MPM and 3 with NSCLC were vaccinated, with 8 patients receiving at least 6 vaccinations; in total, 10 patients were evaluable for immune response. Six out of nine patients tested demonstrated CD4 T-cell proliferation to WT1 specific peptides, and five of the six HLA-A0201 patients tested mounted a CD8 T-cell response. Stimulated T cells were capable of cytotoxicity against WT-1 positive cells. Vaccination also induced polyfunctional CD8 T cell responses.
CONCLUSIONS
This multivalent WT1 peptide analog vaccine induces immune responses in a high proportion of patients with thoracic malignancies with minimal toxicity. A randomized trial testing this vaccine as adjuvant therapy in MPM is planned.
Wilms tumor protein 1 (WT1) is a transcription factor overexpressed in several types of leukemia and solid tumors. For this reason, WT1 is an attractive target for immunotherapy. Four peptide nonamers from WT1 have been identified by others to generate a WT1-specific cytotoxic response in the context of human leukocyte antigen (HLA)-A0201 and A2402. However, as WT1 is a self-antigen, breaking tolerance is a potential obstacle to vaccination. Here, we use a strategy to circumvent tolerance by designing synthetic immunogenic analog peptides that could crossreact to the native peptides (a heteroclitic response). A number of synthetic peptides derived from nonamer sequences of the WT1 protein were designed in which single amino-acid substitutions were introduced at HLA-A0201 major histocompatibility complex (MHC)-binding positions. Several of new peptides could stabilize MHC class I A0201 molecules better than native sequences. Some analogs were also able to elicit WT1-specific T-cell recognition and cytotoxic T-cell lymphocytes more effectively than native sequences. Importantly, T cells stimulated with the new analogs crossreacted with the native WT1 peptide sequence and were able to kill HLA-matched chronic myeloid leukemia cell lines. In conclusion, analog heteroclitic WT1 peptides with increased immunogenicity can be synthesized and are potential cancer vaccine candidates.
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